Enhanced antifungal activity of posaconazole against Candida auris by HIV protease inhibitors, atazanavir and saquinavir

The increasing incidence and dissemination of multidrug-resistant Candida auris represents a serious global threat. The emergence of pan-resistant C. auris exhibiting resistance to all three classes of antifungals magnifies the need for novel therapeutic interventions. We identified that two HIV protease inhibitors, atazanavir and saquinavir, in combination with posaconazole exhibited potent activity against C. auris in vitro and in vivo. Both atazanavir and saquinavir exhibited a remarkable synergistic activity with posaconazole against all tested C. auris isolates and other medically important Candida species. In a time-kill assay, both drugs restored the fungistatic activity of posaconazole, resulting in reduction of 5 and 5.6 log10, respectively. Furthermore, in contrast to the individual drugs, the two combinations effectively inhibited the biofilm formation of C. auris by 66.2 and 81.2%, respectively. Finally, the efficacy of the two combinations were tested in a mouse model of C. auris infection. The atazanavir/posaconazole and saquinavir/posaconazole combinations significantly reduced the C. auris burden in mice kidneys by 2.04- (99.1%) and 1.44-log10 (96.4%) colony forming unit, respectively. Altogether, these results suggest that the combination of posaconazole with the HIV protease inhibitors warrants further investigation as a new therapeutic regimen for the treatment of C. auris infections.

Invasive fungal infections pose an underestimated threat to the public health 1,2 .Fungal infections are on the rise globally, particularly among immunocompromised individuals, reaching over 11 million infections and 1.5 million deaths annually [3][4][5] .The majority of invasive fungal infections is caused by Candida species which are associated with high mortality rates (up to 60%) 2,6 .The emerging fungal species, Candida auris has spread to more than 45 countries, causing serious hospital outbreaks, and significantly contributed to the worldwide health challenge of antifungal resistance 7 .The incidence and dissemination of multidrug-resistant C. auris-related infections are alarmingly growing and are associated with high mortality rates 8 .Thus, there is a desperate need to develop new antifungals for the treatment of multidrug-resistant C. auris infections.
Candida auris possesses an arsenal of virulence factors known to contribute to its pathogenicity.Among these, biofilm formation is a major virulence factor of C. auris which plays an important role in the nosocomial outbreaks of life-threatening invasive candidiasis 9 .Notably, C. auris infections are frequently associated with medical devices such as catheters, where it forms biofilms that adhere to the device surface, leading to device malfunction and serving as a source of bloodstream infection 10 .Furthermore, the formation of biofilms significantly contributes to the escalation of antifungal resistance, as these biofilms enable C. auris cells to tolerate higher concentrations of antifungal agents 10,11 .Consequently, novel therapeutic approaches are urgently needed to effectively manage C. auris infections, particularly those associated with biofilm formation 9 .
Posaconazole, a second-generation azole antifungal, has a potent and broad-spectrum antifungal activity against pathogenic fungi, including C. auris 12 .Posaconazole has a more favorable safety and tolerability profile when compared to the other azoles like fluconazole, itraconazole, and voriconazole.Long-term treatment with posaconazole has demonstrated a reduced risk of inducing side effects 13,14 .Furthermore, posaconazole's extended half-life of 15-35 h, widespread tissue distribution, and gradual elimination contribute to its therapeutic effectiveness 15 .In addition to these attributes, posaconazole offers therapeutic advantages over fluconazole and voriconazole because it is less likely to be affected by mutations in the ergosterol gene (ERG11), which codes for www.nature.com/scientificreports/lanosterol demethylase, the target enzyme of azoles 11 .Furthermore, it was reported that patients infected with fluconazole-resistant C. glabrata or C. tropicalis were able to recover when treated with posaconazole 1 .However, the emergence of pan-resistant C. auris, displaying resistance to all three classes of antifungals, underscores the urgency for the development of new therapeutics 16 .
Previously, we reported that lopinavir exhibited potent synergistic interactions with fluconazole, itraconazole and voriconazole against C. auris 17 .We also identified atazanavir and saquinavir as potent drugs that restored the activity of itraconazole against C. auris in vitro and in vivo 18,19 .Building upon our previous studies, we aimed herein to enhance the activity of posaconazole against C. auris using the HIV protease inhibitors (atazanavir and saquinavir).We also evaluated the activity of the combinations of posaconazole with atazanavir and saquinavir in inhibiting the C. auris biofilm.Finally, we investigated the in vivo efficacy of atazanavir/posaconazole and saquinavir/posaconazole combinations in a mouse model of C. auris infection.

Posaconazole exhibits synergistic antifungal activity with atazanavir and saquinavir in vitro
Checkerboard assay was performed to corroborate the potential synergistic interactions for the combinations atazanavir/posaconazole and saquinavir/posaconazole against 17 C. auris isolates.Atazanavir and saquinavir were found to interact synergistically with posaconazole against 100% of C. auris isolates decreasing the minimum inhibitory concentrations (MICs) of posaconazole by 4-17 folds.Atazanavir/posaconazole and saquinavir/posaconazole combinations demonstrated synergistic fractional inhibitory concentration indices (FICIs) of (0.06-0.28) (Table 1).Additionally, these two combinations showed similar synergistic interactions against other medically important Candida species such as C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata with FICIs ranging between 0.12 and 0.38 (Table 2).

Atazanavir and saquinavir enhance the fungistatic activity of posaconazole against C. auris
To investigate the impact of the two combinations on the growth kinetic of C. auris, a time-dependent killing assay was conducted against C. auris AR0390 for 48 h (Fig. 1).Single drug treatment of either atazanavir (16 µg/ ml), saquinavir (16 µg/ml), or posaconazole (0.25 µg/ml) did not affect the fungal growth over time, where their growth pattern was similar to that of the mock (negative) control.However, the atazanavir/posaconazole combination reduced C. auris colony forming unit (CFU) by 3.78-and 5.02-log 10 as compared to the mock (negative) control, after 24 and 48 h, respectively.On the other hand, saquinavir/posaconazole combination reduced C. auris CFU by 4.19-and 5.62-log 10 as compared to the mock (negative) control, after 24 and 48 h, respectively (Fig. 1A).This was confirmed by the spotting assay which showed that C. auris had exponential growth in the presence and absence of atazanavir (16 µg/ml), saquinavir (16 µg/ml), posaconazole (0.25 µg/ml).Conversely, the growth of C. auris was inhibited significantly in the presence of the two combinations (Fig. 1B).Additionally, we used a posaconazole test strip in RPMI 1640 agar cultured with 10 6 C. auris cells to determine whether HIV protease inhibitors (atazanavir, and saquinavir) improve the activity of posaconazole against azole-resistant C. www.nature.com/scientificreports/auris.As expected, atazanavir and saquinavir (32 µg/ml) decreased the MIC of posaconazole from 1 µg/ml to 0.38 and 0.19 µg/ml (2.6-and 5.2-folds reduction), respectively, against C. auris (Fig. 1C).

Atazanavir/posaconazole and saquinavir/posaconazole combinations inhibit biofilm formation of C. auris
Due to the strong antifungal activity against C. auris, the two combinations were selected to investigate the biofilm inhibition activity.As shown in Fig. 2, atazanavir, and saquinavir, at a low concentration of 16 µg/ml, in combination with sub-inhibitory concentration of posaconazole (0.03 µg/ml) showed a significant inhibition of the C. auris biofilm formation by 66.2%, and 81.2%, respectively, compared to the mock (negative) control.

Atazanavir and saquinavir enhance posaconazole's efficacy in a mouse model of C. auris infection
A mouse model of disseminated C. auris infection was used to investigate the antifungal activity of the two combinations (atazanavir/posaconazole, saquinavir/posaconazole) in vivo.First, the posaconazole dose was optimized to determine the highest dose that does not result in significant reduction in the kidney fungal burden.Mice were treated with different doses of posaconazole (0.25, 0.5, 1, 3, 5 mg/kg) and the optimal dose of posaconazole was determined as 3 mg/kg, which did not result in significant difference as compared to the untreated control (Fig. S1).Next, we tested the in vivo efficacy of the two combinations on this mouse model in the presence of the bioavailability enhancer, ritonavir (to mimic the 3:1 atazanavir: ritonavir and the 10:1 saquinavir: ritonavir ratio used clinically) 20 .The doses chosen of atazanavir/ritonavir (90/30 mg/kg) and saquinavir/ ritonavir (200/20 mg/kg) was equal or lower than the dose required in mice to achieve plasma concentration, which is equivalent to the therapeutic levels in humans 21,22 .Treatments were administered orally for 2 days.The next day, mice were euthanized and the C. auris burden in their kidneys was determined.Neither posaconazole alone, nor the HIV protease inhibitors (atazanavir-ritonavir, or saquinavir-ritonavir) treatments was capable of reducing the fungal burden of C. auris when compared to the untreated control.In contrast, compared to the vehicle control, the posaconazole/atazanavir-ritonavir and posaconazole/saquinavir-ritonavir combinations significantly reduced the burden of C. auris in the murine kidneys, producing 2.04 (99.1%) and 1.44-log 10 CFU (96.4%) reduction.Moreover, when compared to posaconazole treatment, posaconazole/atazanavir-ritonavir and posaconazole/saquinavir-ritonavir combinations generated 1.40 (96%) and 0.8-log 10 CFU (84.2%) reduction, respectively (Fig. 3).

Discussion
Candida auris is a widespread pathogenic yeast causing severe invasive candidiasis in critically ill and immunocompromised patients 23 .Due to its multidrug resistance, severity of infections, and nosocomial outbreaks with high mortality rate, C. auris represent an alarming paradigm shift for Candida infections 24 .Moreover, the Centers for Disease Control and Prevention (CDC) has classified C. auris as the highest level of threat on the most recent report of antibiotic resistance threat in the United States 25 .Thus, a robust response is needed to develop new antifungals and treat such infections.Fluconazole is the mainstay of antifungal therapy for disseminated candidiasis.Nonetheless, posaconazole is recommended as an alternative treatment option for patients who are infected with fluconazole or itraconazole-resistant Candida 26 .Posaconazole was discovered to be more effective than fluconazole, itraconazole, and voriconazole against Candida isolates based on its MIC values because of the long side chain of posaconazole, which increases its binding affinity to the target 27,28 .Therefore, in this study we evaluated the ability of the HIV protease inhibitors to synergize and potentiate the activity of posaconazole against C. auris.
Here, we found that posaconazole interacted synergistically with both HIV protease inhibitors (atazanavir, and saquinavir) against all 17 C. auris isolates.Additionally, the synergistic combinations exhibited a fungistatic activity reducing the C. auris CFU by 5 and 5.6-logs after 48 h as compared to the mock (negative) control in a time kill assay.Furthermore, the synergistic interaction between atazanavir/saquinavir and posaconazole has proven effective against all other clinically important Candida species, providing an additional clinical advantage to these combinations.
Based on these results and our prior findings, it is evident that both atazanavir and saquinavir exhibit more potent synergistic interactions when combined with itraconazole and posaconazole compared to fluconazole and voriconazole.This suggests a potential therapeutic advantage of using HIV protease inhibitors in combination with itraconazole and posaconazole, as mutations in the target gene (ERG11) appear to exert a lesser influence on the binding and activity of these particular antifungal agents 11,18,19 .Biofilm formation is one of the major virulence factors of C. auris 29 .C. auris possesses high ability to form a strong adhesive biofilm on non-living structures, such as indwelling medical devices or implants 10 .Additionally, it was reported that the expression of efflux pump transporters, including both ATP-binding cassette (ABC) and Major facilitator superfamily (MFS), increased during the maturation stage of biofilm formation 3 .HIV protease inhibitors were reported to impair biofilm formation and disrupt the mature biofilm of some fungal species such as C. albicans and Trichosporon 30,31 .Therefore, we investigated the biofilm inhibition activity of atazanavir/ posaconazole and saquinavir/posaconazole combinations.Both combinations significantly inhibited the C. auris biofilm formation.
Finally, we evaluated the synergistic combinations atazanavir/posaconazole and saquinavir/posaconazole in a C. auris disseminated infection mouse model.Posaconazole alone has previously demonstrated its efficacy in numerous mouse models of disseminated candidiasis 32 .However, the in vivo efficacy of posaconazole was not reported against C. auris before.Thus, we were encouraged to test the two combinations against mouse model of C. auris infection.First, we evaluated the efficacy of different doses of posaconazole to determine the optimal dose that does not result in a significant reduction of the fungal burden.We found that posaconazole's dose of 3 mg/kg was the highest dose that did not significantly reduced C. auris CFU as compared to the untreated control.This dose was used in combination with either atazanavir or saquinavir to test the in vivo efficacy of the combinations against C. auris.Ritonavir was added to each combination as a pharmacokinetic enhancer 33,34 .Atazanavir-ritonavir and saquinavir-ritonavir did not reduce the fungal CFU, similar to previous studies 18,19 .On the other hand, the two combinations atazanavir/posaconazole and saquinavir/posaconazole, in the presence of ritonavir, significantly reduced the C. auris CFU burden in mice kidneys, generating 2.04 (99.1%) and 1.44-log 10 CFU (96.4%) reduction, compared to the mock (negative) control group.Remarkably, the in vivo efficacy of the atazanavir/posaconazole combination (with 2.04 -log 10 reduction) surpassed the in vivo efficacy of atazanavir/ itraconazole combination (with 1.15-log 10 reduction) against C. auris 18 .Additionally, the in vivo efficacy of the saquinavir/posaconazole combination (with 1.44-log 10 reduction) surpassed the in vivo efficacy of saquinavir/ itraconazole combination (with 0.85-log 10 reduction) against C. auris 19 .
In conclusion, the atazanavir/posaconazole and saquinavir/posaconazole combinations displayed not only in vitro synergistic antifungal activity but also anti-biofilm activity against C. auris.Moreover, our study found that the combinations efficiently reduced C. auris burden in mice kidneys.Thus, atazanavir and saquinavir can be considered as promising agents for enhancing the potency of posaconazole against azole-resistant C. auris.

Minimum inhibitory concentration and checkerboard assay
The MICs values of posaconazole, the HIV protease inhibitors (atazanavir and saquinavir) were evaluated against C. auris isolates following the CLSI guidelines 35 .The combinations of posaconazole with atazanavir or saquinavir were determined against 17 C. auris isolates using the checkerboard method, as described elsewhere 36 .Similarly, the two combinations were evaluated against other Candida species including C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata.The FICI was calculated and interpreted as follows: FICI of > 4 was classified as antagonism, FICI of > 0.5-4: indifference, and FICI of ≤ 0.5: synergism 37 .To confirm the ability of atazanavir and saquinavir to improve the activity of posaconazole against C. auris, we used posaconazole test strips.C. auris AR0390 was cultured in RPMI 1640 agar in the presence of atazanavir, saquinavir or dimethyl sulfoxide (DMSO).
Next, the posaconazole test strip was added, and the agar plate was incubated at 35 °C for 24 h.The concentration at which a zone of inhibition intercepted with the posaconazole strip used as the MIC.

Time-kill kinetics and spotting assays
A time-kill assay was used to investigate the killing kinetics of the atazanavir/posaconazole and saquinavir/ posaconazole combinations against C. auris AR0390, as described before 17,38,39 .Briefly, exponential phase C. auris cells were diluted to ~ 10 4 CFU/ml in RPMI 1640 medium and incubated with either atazanavir (16 µg/ ml), saquinavir (16 µg/ml), posaconazole (0.25 µg/ml), atazanavir/posaconazole combination, or saquinavir/ posaconazole combination.DMSO-treated cells were used as a negative (mock) control.Aliquots were taken at specific time points (0, 6, 12, 24, 30, 36, and 48 h), diluted and counted to determine the number of viable cells.The data are presented as the average of three independent experiments.For the spotting assay, aliquots from the time point of 24-h were plated onto YPD agar plates and incubated at 35 °C for 24 h before the plates were scanned.

C. auris biofilm formation assay
The C. auris biofilm formation assay was performed as described previously [40][41][42] .To evaluate the effect of the atazanavir/posaconazole and saquinavir/posaconazole combinations in preventing biofilm formation, HIV-protease inhibitors (atazanavir and saquinavir) diluted in RPMI medium (at 16 μg/ml) with and without posaconazole (0.03 μg/ml) were added to 96-well plates containing 10 6 CFU/ml of C. auris AR0390 cells, and the plates were incubated at 35 °C for 24 h.To assess the biofilm inhibition of the C. auris AR0390 isolate, adherent biofilms were stained for 30 min with 100 μl of 0.1% (wt/vol) crystal violet.After crystal violet was removed, the cells were washed 3 times with PBS and the plates were allowed to dry.The resultant biofilm biomasses were then quantified by dissolving the crystal violet-stained biofilms in 100 μl of ethanol before recording absorbance values (OD 600 ).

C. auris infection mouse model
The Virginia Tech Animal Care and Use Committee reviewed and approved the mouse study, which was conducted strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.The mouse studies are in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.To assess the efficacy of the two combinations atazanavir/posaconazole and saquinavir/ posaconazole in vivo against C. auris, we used a mouse model of disseminated C. auris infection as reported previously 43 .First, we conducted a dose-response study for posaconazole to determine the dose that can be used in combination with either atazanavir or saquinavir.Female CD-1 mice were injected intraperitoneally (I.P.) with cyclophosphamide (200 mg/kg) and (150 mg/kg) 4 days and 1 day, respectively before infection.On the infection day, mice were injected I.P with 4.5 × 10 7 cells/mouse of C. auris AR0390.Two hours later, mice were randomly distributed into 6 groups and administered different doses of posaconazole (0.25, 0.5, 1, 3, 5 mg/kg) orally once daily for two days.One day later, mice were humanely euthanized using CO 2 , and their kidneys were extracted, homogenized, serially diluted, and plated onto YPD agar supplemented with chloramphenicol.YPD plates we then incubated for 24 h at 35 °C for the CFU determination.We determined the optimal dose of posaconazole as 3 mg/kg to be used in the following experiment.Next, we assessed the in vivo efficacy of the two combinations, with adding ritonavir as pharmacokinetic enhancer.Mice were rendered neutropenic by cyclophosphamide injection as described above.On the challenge day, mice were injected I.P. with 5.67 × 10 7 cells/mouse of C. auris AR0390.Two hours after infection, mice were randomly allocated into groups and all treatments were given orally as follows: (A) Control, (B) posaconazole (3 mg/kg), (C) atazanavir-ritonavir (90-30 mg/kg), D) saquinavir-ritonavir (200-20 mg/kg), (E) posaconazole/ atazanavir-ritonavir (3/90-30 mg/kg), (F) posaconazole/saquinavir-ritonavir (3/200-20 mg/kg).In these groups, posaconazole was administered once daily while atazanavir and saquinavir were administered with ritonavir twice daily for 2 days.Twelve-hours after the last dose, mice were euthanized and the C. auris burden in the mice kidneys was determined as described above.The data was analyzed via one-way analysis of variance (ANOVA) with post-hoc Dunnett's test for multiple comparisons.

Statistical analyses
The statistical tests and significance were determined and indicated in each figure legend using GraphPad Prism 8 software.

Figure 1 .
Figure 1.Time-kill of the HIV protease inhibitors (atazanavir (ATV), saquinavir (SQV)), and posaconazole (POS) alone and in combination against C. auris.(A) Time kill curve of C. auris AR0390.Data are presented as average log 10 CFU/ml of C. auris AR0390 at the corresponding time points.Statistical difference was measured via two-way analysis of variance (ANOVA).An asterisk (*) denotes a statistically significant difference (P < 0.0001) from the POS-treated cells.(B) Spotting assay was used to visualize the growth kinetics of C. auris in the presence and absence of treatments at the 24-h time point.(C) Effects of atazanavir and saquinavir on azole-resistant C. auris.MIC test strips of posaconazole (ranged from 32-0.002 µg/ml) in the presence and absence of atazanavir and saquinavir at 32 µg/mL.